Name: injecting gryllus bimaculatus eggs
Uploaded: 2019-08-22T13:00:00
Description: Watch this Scientific Journal Video about Injecting Gryllus bimaculatus Eggs at JoVE.com

Research reported in this project was supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P20GM10342 to HH3, and by NSF award number IOS-1257217 to CGE.

1. Hardware Setup and Preparation of Materials
NOTE: Please see Table 1 and Table of Materials for preparation of solutions, reagent, and equipment details.
<ol>
	<li>Set up a dissecting microscope in order to see eggs and guide the injection needle. (Figure 1A shows a dissecting microscope equipped with fluorescence.) Fluorescence capability is advantageous but not necessary.</li>
	<li>Position a 3-axis microma...

<ol>
	<li>Abzhanov, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Are+we+there+yet?+Tracking+the+development+of+new+model+systems.">Are we there yet? Tracking the development of new model systems.</a> Trends in Genetics. 24 (7), 353-360 (2008).</li><li>Krebs, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+August+Krogh+principle:+&#8220;For+many+problems+there+is+an+animal+on+which+it+can+be+most+conveniently+studied.">The August Krogh principle: &#8220;For many problems there is an animal on which it can be most conveniently studied.</a> Journal of Experimental Zoology Part B: Molecular and Developmental Evolution. 194 (1), 221-226 (1975).</li><li>Krogh, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Progress+of+Physiology.">The Progress of Physiology.</a> American Journal of Physiology. 90 (2), 243-251 (1929).</li><li>Huber, F., Moore, T. E., Loher, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Cricket neurobiology and behavior. , (1989).</li><li>Horch, H. W., Mito, T., Popadic, A., Ohuchi, H., Noji, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Cricket as a Model Organism: Development, Regeneration, and Behavior. , (2017).</li><li>Misof, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phylogenomics+resolves+the+timing+and+pattern+of+insect+evolution.">Phylogenomics resolves the timing and pattern of insect evolution.</a> Science. 346 (6210), 763-767 (2014).</li><li>Nakamura, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+of+transgenic+cricket+embryos+reveals+cell+movements+consistent+with+a+syncytial+patterning+mechanism.">Imaging of transgenic cricket embryos reveals cell movements consistent with a syncytial patterning mechanism.</a> Current Biology. 20 (18), 1641-1647 (2010).</li><li>Shinmyo, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=piggyBac-mediated+somatic+transformation+of+the+two-spotted+cricket,+Gryllus+bimaculatus.">piggyBac-mediated somatic transformation of the two-spotted cricket, Gryllus bimaculatus.</a> Development, growth &amp; differentiation. 46 (4), 343-349 (2004).</li><li>Watanabe, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-transgenic+genome+modifications+in+a+hemimetabolous+insect+using+zinc-finger+and+TAL+effector+nucleases.">Non-transgenic genome modifications in a hemimetabolous insect using zinc-finger and TAL effector nucleases.</a> Nature communications. 3, 1017 (2012).</li><li>Wang, H., La Russa, M., Qi, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPR/Cas9+in+Genome+Editing+and+Beyond.">CRISPR/Cas9 in Genome Editing and Beyond.</a> Annual Review of Biochemistry. 85 (1), 227-264 (2016).</li><li>Awata, H., Watanabe, T., Hamanaka, Y., Mito, T., Noji, S., Mizunami, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Knockout+crickets+for+the+study+of+learning+and+memory:+Dopamine+receptor+Dop1+mediates+aversive+but+not+appetitive+reinforcement+in+crickets.">Knockout crickets for the study of learning and memory: Dopamine receptor Dop1 mediates aversive but not appetitive reinforcement in crickets.</a> Scientific Reports. 5, 15885 (2015).</li><li>Horch, H. W., Liu, J. J., Mito, T., Popadic, A., Watanabe, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protocols+in+the+Cricket.">Protocols in the Cricket.</a> The Cricket as a Model Organism: Development, Regeneration, and Behavior. , 327-370 (2017).</li><li>Watanabe, T., Noji, S., Mito, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome+Editing+in+the+Cricket,+Gryllus+bimaculatus.">Genome Editing in the Cricket, Gryllus bimaculatus.</a> Genome Editing in Animals. , 219-233 (2017).</li><li>Kainz, F., Ewen-Campen, B., Akam, M., Extavour, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Notch/Delta+signalling+is+not+required+for+segment+generation+in+the+basally+branching+insect+Gryllus+bimaculatus.">Notch/Delta signalling is not required for segment generation in the basally branching insect Gryllus bimaculatus.</a> Development. 138 (22), 5015-5026 (2011).</li><li>Miyawaki, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Involvement+of+Wingless/Armadillo+signaling+in+the+posterior+sequential+segmentation+in+the+cricket,+Gryllus+bimaculatus+(Orthoptera),+as+revealed+by+RNAi+analysis.">Involvement of Wingless/Armadillo signaling in the posterior sequential segmentation in the cricket, Gryllus bimaculatus (Orthoptera), as revealed by RNAi analysis.</a> Mechanisms of Development. 121 (2), 119-130 (2004).</li><li>Donoughe, S., Kim, C., Extavour, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+live-imaging+of+embryos+in+microwell+arrays+using+a+modular+specimen+mounting+system.">High-throughput live-imaging of embryos in microwell arrays using a modular specimen mounting system.</a> Biology Open. 7 (7), bio031260 (2018).</li><li>Donoughe, S., Nakamura, T., Ewen-Campen, B., Green, D. A., Henderson, L., Extavour, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BMP+signaling+is+required+for+the+generation+of+primordial+germ+cells+in+an+insect.">BMP signaling is required for the generation of primordial germ cells in an insect.</a> Proceeding of the National Academy of Science USA. 111 (11), 4133-4138 (2014).</li><li>Larson, E., Andres, J., Harrison, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+the+male+ejaculate+on+post-mating+prezygotic+barriers+in+field+crickets.">Influence of the male ejaculate on post-mating prezygotic barriers in field crickets.</a> PLOS ONE. 7 (10), e46202 (2012).</li><li>Donoughe, S., Extavour, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+development+of+the+cricket+Gryllus+bimaculatus.">Embryonic development of the cricket Gryllus bimaculatus.</a> Developmental Biology. , (2016).</li><li>Matsuoka, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short+germ+insects+utilize+both+the+ancestral+and+derived+mode+of+Polycomb+group-mediated+epigenetic+silencing+of+Hox+genes.">Short germ insects utilize both the ancestral and derived mode of Polycomb group-mediated epigenetic silencing of Hox genes.</a> Biology Open. 4 (6), 702-709 (2015).</li><li>Rosenberg, M., Lynch, J., Desplan, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heads+and+tails:+Evolution+of+antero-posterior+patterning+in+insects.">Heads and tails: Evolution of antero-posterior patterning in insects.</a> Biochimica et biophysica acta. 1789 (4), 333-342 (2009).</li><li>Bacon, J., Strausfeld, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonrandom+resolution+of+neuron+arrangements.">Nonrandom resolution of neuron arrangements.</a> Neuroanatomical Techniques: Insect Nervous System. , 357-372 (1980).</li></ol>

The two main challenges with this technique are the related issues of optimal needle size and survivability. Though smaller needles improve survivability, needles with narrower lumens have a greater degree of capillary forces at work, which makes it more likely that yolk will move into the needle causing it to clog. In the best case, blockages can be cleared simply by injecting another egg or by clearing the needle as described above. One can also attempt to increase the balance pressure on the microinjector until the yo...

The ability to modify the genome or influence gene expression in organisms is the basis for the design of many types of experiments testing functional causality. It is also critical for the comparative and evolutionarily-relevant work that genomic and non-genomic modification techniques be available in organisms outside traditional genetic laboratory animal model systems (e.g., Mus musculus, Danio rerio, Drosophila melanogaster, and Caenorhabditis elegans). Whether it is the desire to understand organismal diversity1 or one&#39;s adherence to Krogh&#39;s principle, that for every biological question there is an organism best suited to its solution2,3, the ability to modify genomes or influence the gene expression is essential for modern experimental designs.
The cricket Gryllus bimaculatus is an emerging model system. Used for the last century in neuroethology experiments4, the last two decades have witnessed an increased experimental interest in the cricket, particularly focused on the evolution and development of this organism5. The cricket is a hemimetabolous insect that branches basally to well-studied holometabolous insects, such as D. melanogaster and Tribolium castaneum6. Due to its useful position on the evolutionary tree, scientists are interested in asking modern, sophisticated experimental questions in this insect, which has led to a growing interest in adapting molecular tools for the use in G. bimaculatus.
Injections of molecular reagents into cricket eggs can be used for genomic modification experiments as well as non-genomic manipulations of gene expression in embryos. For example, transgenic G. bimaculatus carrying eGFP insertions have been created using the transposase piggyBac7,8. Investigators have successfully created knockout G. bimaculatus using Zinc-finger nucleases (ZFNs) and transcription activator-like (TAL) effector nucleases (TALENs) to introduce double-stranded breaks in specific genomic regions9. Though ZFNs and TALENs allow site-specific targeting in animals beyond the big four model systems, these reagents have quickly been surpassed by the CRISPR/Cas9 system, which is simpler to use, more efficient, and highly flexible10. CRISPR has been used in G. bimaculatus to produce knock-out11 as well as knock-in lines12,13 In addition to genomic modification, dsRNA can be injected into eggs to knock down mRNA expression in developing embryos, allowing investigators to understand the role of specific transcripts throughout development14,15. Some limited details on how to inject cricket eggs have been published previously12.
Here, we describe a detailed protocol for injecting early G. bimaculatus eggs. This protocol is effective and easily adaptable to various laboratory settings, injection materials, and possibly to other insects. While additional details for designing and implementing genomic modification and knockdown experiments have been published elsewhere12,13, these approaches will ultimately rely on the injection protocol detailed here.

<table>
	<tbody>
		<tr>
			<td>Fluorescent dissecting microscope</td>
			<td>Leica</td>
			<td>M165 FC</td>
			<td>Stereomicroscope with fluorescence</td>
			<td></td>
		</tr>
		<tr>
			<td>External light source for fluorescence</td>
			<td>Leica</td>
			<td>EL 6000</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microinjector</td>
			<td>Narishige</td>
			<td>IM-300</td>
			<td>-Accessories may include Injection Needles Holder, Input Hose (with a hose connector), AC Power Cord, Foot Switch, Silicone Rubber Gasket-</td>
			<td></td>
		</tr>
		<tr>
			<td>mCherry filter cube</td>
			<td>Leica</td>
			<td>M205FA/M165FC</td>
			<td>Filter cube for mCherry or similar red dye will work</td>
			<td></td>
		</tr>
		<tr>
			<td>Micromanipulator</td>
			<td>World Precision Instruments, Inc.</td>
			<td>M3301R</td>
			<td>Used with Magnetic Stand (Narishige, Type GJ-8)</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic stand</td>
			<td>Narishige</td>
			<td>MMO-202ND</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette Holder (Needle holder)</td>
			<td>Narishige</td>
			<td>HD-21</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="2">Tubing to connect air source to microinjector</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Egg well stamp</td>
			<td>3D printed</td>
			<td>custom</td>
			<td>3D printed on a Lulzbot Taz 5 using Poly Lactic Acid thermoplastic</td>
			<td></td>
		</tr>
		<tr>
			<td>Microwave</td>
			<td>various</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator or temperature controlled room</td>
			<td>various</td>
			<td></td>
			<td>Temperatures of 23.5-26&#176;C are needed.</td>
			<td></td>
		</tr>
		<tr>
			<td>cricket food</td>
			<td>various</td>
			<td></td>
			<td>cat food or fish flakes are appropriate food.&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>cricket wter</td>
			<td>vairous</td>
			<td></td>
			<td>Water can be held in vials and presented to crickets through cotton balls</td>
			<td></td>
		</tr>
		<tr>
			<td>cricket shelter</td>
			<td>arious</td>
			<td></td>
			<td>Shelter materials can include crumpled paper towels or egg cartons</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass capillary tubes</td>
			<td>World Precision Instruments, Inc.</td>
			<td>Item no. 1B100F-4</td>
			<td>Kwik-Fil&#8482; Borosilicate Glass Capillaries, 100mm length, 0.58 mm ID, 1.0 mm OD, with filament</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette puller</td>
			<td>Flaming/Brown</td>
			<td>Model P-97</td>
			<td>Distributed by Sutter Instrument Co.</td>
			<td></td>
		</tr>
		<tr>
			<td>Beveller/Micro grinder</td>
			<td>Narishige</td>
			<td>Model EG-45/EG-400</td>
			<td>EG-400 includes a microscope head</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dishes</td>
			<td>CellTreat</td>
			<td>Product code 229693</td>
			<td>90mm diameter</td>
			<td></td>
		</tr>
		<tr>
			<td>Play Sand</td>
			<td>Sandtastik Products Ltd.</td>
			<td>B003U6QLVS</td>
			<td>White play sand</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>American Bioanalytical</td>
			<td>AB000972</td>
			<td>Agarose GPG/LE ultrapure</td>
			<td></td>
		</tr>
		<tr>
			<td>Egg Strainer: Extra Fine Twill Mesh Stainless Steel Conical Strainers</td>
			<td>US Kitchen Supply</td>
			<td>Model SS-C123</td>
			<td>Pore size should be between 0.5 - 1.0 mm</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin Streptomycin</td>
			<td>Gibco by Life Technologies</td>
			<td>Ref 15070-063</td>
			<td>Pen Strep</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic tweezers</td>
			<td>Sipel Electronic SA</td>
			<td>P3C-STD</td>
			<td>Black Static Dissipative, 118mm</td>
			<td></td>
		</tr>
		<tr>
			<td>syringe filters, 25mm diameter, 0.45 &#181;m</td>
			<td>Nalgene</td>
			<td>725-2545</td>
			<td>Use with 1 ml syringe</td>
			<td></td>
		</tr>
		<tr>
			<td>1 mL syringe, with Tuberculin Slip Tip</td>
			<td>Becton Dickinson</td>
			<td>309602</td>
			<td>Use with syring filter to filter Injection Buffer , Luer-Lok tip syringes would also work</td>
			<td></td>
		</tr>
		<tr>
			<td>Air tank (optional)</td>
			<td>Midwest Products</td>
			<td>Air Works&#174;</td>
			<td>Portable air tank</td>
			<td></td>
		</tr>
		<tr>
			<td rowspan="2">Rhodamine dye</td>
			<td rowspan="2">Thermofisher</td>
			<td rowspan="2">D-1817</td>
			<td rowspan="2">dextran, tetramethylrhodamine 10,000MW,</td>
			<td></td>
		</tr>
		<tr>
			<td>20 mL loading tips</td>
			<td>Eppendorf</td>
			<td>Order no. 5242 956.003</td>
			<td>epT.I.P.S. 20uL Microloader</td>
			<td></td>
		</tr>
		<tr>
			<td>Compound microscope</td>
			<td>Zeiss</td>
			<td>Axioskope 2 plus</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>20X objective</td>
			<td>Ziess</td>
			<td>Plan-Apochromat 20x/0.75 M27</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>camera</td>
			<td>Leica</td>
			<td>DMC 5400</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Leica Application Suite&#160; software</td>
			<td>Leica</td>
			<td>LAS</td>
			<td>Version 4.6.2 used here</td>
			<td></td>
		</tr>
	</tbody>
</table>

injecting gryllus bimaculatus eggs

Altering gene function in a developing organism is central to different kinds of experiments. While tremendously powerful genetic tools have been developed in traditional model systems, it is difficult to manipulate genes or messenger RNA (mRNA) in most other organisms. At the same time, evolutionary and comparative approaches rely on an exploration of gene function in many different species, necessitating the development and adaptation of techniques for manipulating expression outside currently genetically tractable species. This protocol describes a method for injecting reagents into cricket eggs to assay the effects of a given manipulation on embryonic or larval development. Instructions for how to collect and inject eggs with beveled needles are described. This relatively straightforward technique is flexible and potentially adaptable to other insects. One can gather and inject dozens of eggs in a single experiment, and survival rates for buffer-only injections improve with practice and can be as high as 80%. This technique will support several types of experimental approaches including injection of pharmacological agents, in vitro capped mRNA to express genes of interest, double-stranded RNA (dsRNA) to achieve RNA interference, use of clustered regularly interspaced short palindromic repeats (CRISPR) in concert with CRISPR-associated protein 9 (Cas9) reagents for genomic modification, and transposable elements to generate transient or stable transgenic lines.

Crickets readily lay eggs in the moist material, and providing adequate material, such as moist sand or dirt, induces them to lay a large number of eggs. This is especially effective if crickets are first deprived of egg-laying material for 8&#8211;10 h. Eggs laid in clean sand can be easily separated, collected (Figure 1B) and placed into custom-designed egg wells for injection (Figure 1C). A dissecting microscope, a microinject...

Watch this Scientific Journal Video about Injecting Gryllus bimaculatus Eggs at JoVE.com

Injecting Gryllus bimaculatus Eggs

Here we present a protocol to inject cricket eggs, a technique which serves as a foundational method in many experiments in the cricket, including, but not limited to, RNA interference and genomic manipulation.

Injecting Gryllus bimaculatus Eggs

Altering gene function in a developing organism is central to different kinds of experiments. While tremendously powerful genetic tools have been ...

This technique serves as a foundational method for experiments designed to assay the embryonic or larval development of the cricket. The main advantage of this technique is that it is flexible and will support several types of experimental approaches, such as those utilizing RNA interference or genomic manipulation. Demonstrating the procedure will be Hadley Wilson Horch, the principal investigator in the laboratory.To begin this procedure wet the spinning grinder plane of the beveler with dripping water. Place the pulled needle into the beveler and turn it to a 20 degree angle. Lower the needle until it just touches the grinding plate and bevel it for two to three minutes.Assess the bevel by placing the beveled needle on double stick tape that has been adhered to a glass microscope slide. Place the slide on the stage of a compound microscope equipped with a camera and image acquisition software. Acquire an image of the needle using a 20x objective.Use the imaging software to measure the interior lumen diameter of the needle just proximal to the beveled opening. Discard any needles with an opening smaller than eight micrometers or larger than 12 micrometers. First, make 40 milliliters of 1%agarose in water and pour it into a 10 centimeter Petri dish.Place an egg well stamp on the surface of the agarose before it solidifies. Once the agarose is solidified, remove the stamp to reveal the wells. Next, fill the agarose well plate with HBS containing 1%penicillin streptomycin.Place the lid on the dish, wrap it in Parafilm and store it at four degrees Celsius. After this, fill a 35 millimeter Petri dish with white playground sand to make an egg collection dish for the crickets to lay eggs in. Cover the egg dish with a paper towel square cut to approximately 18 by 18 centimeters.Fill with tap water through the paper towel. Then, tilt the Petri dish and gently squeeze the top to remove excess water. Tuck the corners of the paper towel square under the dish and place the egg dish in an inverted lid which will help keep the paper towel in place.Place the egg dish into the cricket bin and allow the adult females to oviposit the eggs for one to two hours. Meanwhile, allow the agarose dish to warm to room temperature in preparation for holding the eggs for injection. When ready, remove the egg collection dish, now containing freshly laid eggs, from the cricket bin.Remove the paper towel and place a strainer with a pore size between 0.5 and one millimeters over a beaker. Rinse the contents of the egg laying dish into the strainer under gently running tap water. The sand grains will fall through the strainer mesh into the beaker below while the cricket eggs remain in the strainer basket.Fill a container with reverse osmosis water and place it in a tray. Invert the strainer over the container and tap it against the dish to dislodge the eggs into the water. The eggs will sink to the bottom of the container.Next, use scissors to cut the tip off a P1000 pipette tip to make an opening approximately three millimeters in diameter. Place this tip on a P1000 pipetter, and use it to transfer the eggs from the container to the agarose egg molds dish. Using plastic tweezers, line up the eggs in the agarose wells.Each egg will sink to the bottom of an individual well. Cover the Petri dish with a lid until ready to inject. To begin, place the dish of eggs under the dissecting microscope and select a low magnification of around 10x.Draw up 1.5 microliters of injection solution using 20 microliter loading tips and a P10 pipette and insert the loading tip into the wide end of the injection needle. Expel the injection solution into the injection needle. Insert the needle into the injection housing and tighten, making sure that the needle is properly and firmly inserted into the housing.Then carefully insert the injection housing into the micromanipulator, being careful to not break or be injured by the needle. While looking at both the eggs and needle through the microscope, move the needle near the X in the top left corner of the dish grid. Lower the needle until the tip enters the HBS plus penicillin streptomycin buffered solution in the dish.Center the needle in the field of vision and move the egg dish so that the needle is a few millimeters closer to the edge of the dish than the eggs. After this, set the microscope to the filter appropriate for Rhodamine to observe the fluorescence in the needle and focus on its tip. On the microinjector slowly turn the balance knob clockwise until the injection solution starts to leak out of the needle into the suspension solution.Then turn the knob back counterclockwise slightly until the dye just stops leaking out of the needle. With the needle still centered in the field of view, move the egg dish so that the needle is aimed at the egg to be injected first. Adjust the magnification to about 50x so that a single egg fills most of the field of view.Use both the micromanipulator and one's hand on the egg dish to move the needle into position for the injection. Use the micromanipulator to advance the needle and insert the tip into the first egg to be injected, making sure to insert the needle at 20 to 30%of the egg length from the posterior end of the egg perpendicular to the long axis of the egg. Inject the solution with either the injection foot pedal or the injection button on the microinjector.A small bolus of fluorescent material inside the egg will indicate a successful injection. After this, retract the needle from the egg. If the egg is unintentionally lifted out of its well upon retraction of the needle, use small forceps to push the egg back into the well while retracting the needle.In this study, cricket eggs are injected using a technique that serves as a foundational method in many experiments, including but not limited to, RNA interference and genomic manipulation. The survival rate is one important parameter for assessing the success of this experiment. Most dead eggs can easily be identified by sight as the yolk and embryonic tissue within the egg begin to clump unevenly, and eventually most of the yolk will migrate to one side of the egg.When assessed after four to six days, greater than 85%of uninjected eggs survived, while eggs injected with experimental reagents generally had a lower survival rate. Controlling for all aspects of the injection itself, including the needle puncture, the introduction of dye and buffer, or the presence of vehicle or double stranded RNA, is required in order to understand the true effects of the experimental manipulation attempted. The way one assesses the phenotypic results of injection depends on what was injected.For example, phenotypic changes as a result of specific mRNA knockdowns may be obvious at the gross anatomy level. If on the other hand a cDNA encoding an EGFP tagged histone 2B gene under the control of a G.bimaculatus actin promoter is inserted into the cricket genome using piggyback transposase, it becomes possible to visualize each nucleus in the egg using fluorescence to excite the EGFP tagged hist 2B protein. Adjusting the balance so that the needles don't clog is a critical step in this procedure.You may need to make several adjustments after subsequent injections since yolk sometimes clogs the needle tip. The cricket is a hemimetabolous insect that branches basally to well studied holometabolous insects such as Drosophila. Studying cricket development will help us understand more about evolution and development across the animal kingdom.This technique takes some practice. We recommend practicing control injections until the survival rate four or five days after injection is at least 80%

Research

JoVE Journal

Developmental Biology

Salinity-dependent Toxicity Assay of Silver Nanocolloids Using Medaka Eggs

Salinity is an important characteristic of the aquatic environment. For aquatic organisms it defines the habitats of freshwater, brackish water, and seawater. Tests of the toxicity of chemicals and assessments of their ecological risks to aquatic organisms are frequently performed in freshwater, but the toxicity of chemicals to aquatic organisms depends on pH, temperature, and salinity. There is no method, however, for testing the salinity dependence of toxicity to aquatic organisms. Here, we used medaka (Oryzias latipes) because they can adapt to freshwater, brackish water, and seawater. Different concentrations of embryo-rearing medium (ERM) (1x, 5x, 10x, 15x, 20x, and 30x) were employed to test the toxicity of silver nanocolloidal particles (SNCs) to medaka eggs (1x ERM and 30x ERM have osmotic pressures equivalent to freshwater and seawater, respectively). In six-well plastic plates, 15 medaka eggs in triplicate were exposed to SNCs at 10 mg/L&#8722;1 in different concentrations of ERM at pH 7 and 25 &#176;C in the dark.
We used a dissecting microscope and a micrometer to measure heart rate per 15 sec and eye diameter on day 6 and full body length of the larvae on hatching day (section 4). The embryos were observed until hatching or day 14; we then counted the hatching rate every day for 14 days (section 4). To see silver accumulation in embryos, we used inductively coupled plasma mass spectrometry to measure the silver concentration of test solutions (section 5) and dechorionated embryos (section 6).The toxicity of the SNCs to medaka embryos obviously increased with increasing salinity. This new method allows us to test the toxicity of chemicals in different salinities.

Embryonic stages are the most susceptible to xenobiotics. Although chemical toxicity depends on salinity, no method exists to test the salinity dependence of toxicity to aquatic organisms. Here, we describe a new and high-throughput method for determining the salinity dependence of toxicity to aquatic embryos.

Salinity is an important characteristic of the aquatic environment. For aquatic organisms it defines the habitats of freshwater, brackish water, and ...

In Ovo Feeding of Commercial Broiler Eggs: An Accurate and Reproducible Method to Affect Muscle Development and Growth

Within the past three decades, red meat and poultry scientists focused on developing strategies and technologies to manipulate muscle development during embryonic and fetal development. This area continues to be an area of focus because muscle fiber number is established during this time and determines the basis for all future growth. In poultry, numerous studies demonstrated in ovo feeding of growth factors, vitamins, or other nutrients improved chick embryonic muscle and intestinal development. Improving in ovo muscle development could benefit the poultry industry by possibly influencing meat yield, growth rate, or myopathy conditions. During the past five years, the Gonzalez Laboratory at the University of Georgia developed a nicotinamide riboside in ovo feeding methodology for broiler-chicken embryos, which altered&#160;muscle development. When injected into a developing embryo&#39;s yolk sac, nicotinamide riboside increased pectoralis major muscle weight and muscle fiber density at hatch. This protocol will demonstrate a methodology to accurately and reproducibly conduct in ovo feeding studies utilizing commercial standard- and high-yielding broiler embryos. These data and methods will allow other research groups to perform in ovo feeding studies with much success and reproducibility.

In Ovo Feeding of Commercial Broiler Eggs: An Accurate and Reproducible Method to Affect Muscle Development and Growth

A robust methodology has been developed for conducting in ovo feeding research trials utilizing unincubated commercial broiler eggs to test the ability of natural and synthetic compounds, in this case, nicotinamide riboside, to influence muscle development and growth.

Within the past three decades, red meat and poultry scientists focused on developing strategies and technologies to manipulate muscle development during ...

Investigating Development with Optogenetic Signaling Manipulation in Zebrafish

Optogenetic Signaling Activation in Zebrafish Embryos

Signaling pathways orchestrate fundamental biological processes, including development, regeneration, homeostasis, and disease. Methods to experimentally manipulate signaling are required to understand how signaling is interpreted in these wide-ranging contexts. Molecular optogenetic&#160;tools can provide reversible, tunable manipulations of signaling pathway activity with a high degree of spatiotemporal control and have been applied in vitro, ex vivo, and in vivo. These tools couple light-responsive protein domains, such as the blue light homodimerizing light-oxygen-voltage sensing (LOV) domain, with signaling effectors to confer light-dependent experimental control over signaling. This protocol provides practical guidelines for using the LOV-based bone morphogenetic protein (BMP) and Nodal signaling activators bOpto-BMP and bOpto-Nodal in the optically accessible early zebrafish embryo. It describes two control experiments: A quick phenotype assay to determine appropriate experimental conditions, and an immunofluorescence assay to directly assess signaling. Together, these control experiments can help establish a pipeline for using optogenetic tools in early zebrafish embryos. These strategies provide a powerful platform to investigate the roles of signaling in development, health, and physiology.

Author Spotlight: Manipulating Signaling in Zebrafish Embryos to Decode Cell Fate Decisions 

Optogenetic manipulation of signaling pathways can be a powerful strategy to investigate how signaling is decoded in development, regeneration, homeostasis, and disease. This protocol provides practical guidelines for using light-oxygen-voltage sensing domain-based Nodal and bone morphogenic protein (BMP) signaling activators in the early zebrafish embryo.

Signaling pathways orchestrate fundamental biological processes, including development, regeneration, homeostasis, and disease. Methods to experimentally ...

Enhancing Protein Visualization with Antibody-Mediated Live Imaging

Visualizing Low-Abundance Proteins and Post-Translational Modifications in Living Drosophila Embryos via Fluorescent Antibody Injection

Visualization of proteins in living cells using GFP (Green Fluorescent Protein) and other fluorescent tags has greatly improved understanding of protein localization, dynamics, and function. Compared to immunofluorescence, live imaging more accurately reflects protein localization without potential artifacts arising from tissue fixation. Importantly, live imaging enables quantitative and temporal characterization of protein levels and localization, crucial for understanding dynamic biological processes such as cell movement or division. However, a major limitation of fluorescent tagging approaches is the need for sufficiently high protein expression levels to achieve successful visualization. Consequently, many endogenously tagged fluorescent proteins with relatively low expression levels cannot be detected. On the other hand, ectopic expression using viral promoters can sometimes lead to protein mislocalization or functional alterations in physiological contexts. To address these limitations, an approach is presented that utilizes highly sensitive antibody-mediated protein detection in living embryos, essentially performing immunofluorescence without the need for tissue fixation. As proof of principle, endogenously GFP-tagged Notch receptor that is barely detectable&#160;in living embryos can be successfully visualized after antibody injection. Furthermore, this approach was adapted to visualize post-translational modifications (PTMs) in living embryos, allowing the detection of temporal changes in tyrosine phosphorylation patterns during early embryogenesis and revealing a novel subpopulation of phosphotyrosine (p-Tyr) underneath apical membranes. This approach can be modified to accommodate other protein-specific, tag-specific, or PTM-specific antibodies and should be compatible with other injection-amenable model organisms or cell lines. This protocol opens new possibilities for live imaging of low-abundance proteins or PTMs that were previously challenging to detect using traditional fluorescent tagging methods.

Author Spotlight: Unveiling the Dynamics of Mechanical and Biochemical Signals in Animal Morphogenesis Research 

This protocol describes the customized antibody-based fluorescence labeling and injection into early Drosophila embryos to enable live imaging of low-abundance proteins or post-translational modifications that are challenging to detect using traditional GFP/mCherry-tag approaches.

Visualization of proteins in living cells using GFP (Green Fluorescent Protein) and other fluorescent tags has greatly improved understanding of protein ...

Injecting bOpto-BMP&#47;Nodal Constructs into Zebrafish Embryos 

Injecting bOpto-BMP/Nodal Constructs into Zebrafish Embryos   

To begin, use an incubator and an LED Microplate Illuminator to create a light box for controlling light exposure and temperature. Then, position a six well plate on the upper shelf of the incubator, and check whether the light beam evenly encompasses it. Use a sheet of paper to visualize the distribution of light coverage.Use a light meter to measure irradiance and spatial uniformity. At least one day before injections, make injection dishes and agarose-coated six well plates. Rinse an injection dish mold with embryo medium, and place gently onto molt and agarose.Once the agarose has solidified, delicately use the tab to remove the mold. Next, prepare flamed glass pipettes for immunofluorescence experiments on dechlorinated embryos. Place the end of a glass pipette into a bun and burner flame, and rotate continuously until the edges become smooth.Prepare an injection mix using the mRNAs shown here. For the phenotyping assay, introduce the needle through the corion directly into the center of the cell at the one cell stage, and inject one nanoliter of the injection mix. If conducting an immunofluorescence assay, target the center of the cell of coated embryos at the one cell stage.For both assays, prepare enough embryos for the unexposed non-injected, unexposed injected, exposed non-injected, and exposed injected embryos. Select at least 30 embryos per condition. For the immunofluorescence assay, prepare an additional dish containing non-injected embryos as a proxy to assess the progression of developmental stages.After injection, incubate the embryos in Petri dishes at 28 degrees Celsius.